Nuclear Magnetic Resonance (NMR) is one of the key methods for the determination of molecular structure. This method relies on detecting magnetization resulting from a Boltzmann population difference between the energy levels of nuclear spins that are split by the Zeeman and other magnetic interactions. Under currently attainable magnetic fields of up to ca. 15-23 T and typical temperatures of 100-300 K for NMR experiments, the nuclear spin population difference is small, thus limiting the magnitude of the NMR signal and the range of molecular systems that can be studied by this method. The nuclear spin polarization can be enhanced by up to several orders of magnitude by transferring an intrinsically larger polarization from a magnetically coupled electronic spin system. Such a transfer is achieved by the Dynamic Nuclear Polarization (DNP) method, with applications in the areas of magnetic resonance-based protein structure determination,1-7 imaging,8-11 and materials and surface science.12-15 
In a typical DNP NMR experiment, electromagnetic radiation of the micrometer to millimeter wave length (mm-wave) is applied to saturate the electronic spin transitions of paramagnetic agents that are typically added to the system.16-20 The 660-fold higher polarization of the electronic spins is then partially transferred to the nuclear spins, resulting in a hyperpolarized state for the latter. While the initial high field DNP-NMR studies employed a stable monoradical nitroxide, TEMPO (2,2,6,6-Tetramethyl-1-piperidinyloxy), as a polarizing agent,20 it was later recognized that the use of biradicals facilitates spin polarization transfer at 80-120 K via cross effect mechanism21,22 yielding a greater polarization of the nuclear spins and, thus, an increase in NMR signal. Following the pioneering work by Hu et al. who have tethered a pair of TEMPO monoradicals with a polyethylene glycol linker,23 many bi- or higher order radicals of different geometries and solubility have been synthesized and tested for DNP.24-32 For example, significant improvements in DNP enhancement have been demonstrated by using bTbK25 and its derivatives27,29 that contain two rigidly linked TEMPO fragments to achieve approximate orthogonality of the electronic g-tensors. Recently, bis-cyclohexyl-TEMPO-bisketal (bCTbK) and a higher molecular weight TEKPoL have been shown to retain the DNP enhancement at higher temperatures.31 Two biradicals, TOTAPOL24 and AMUPol28 offer the best compromise between the magnitude of the DNP enhancement and solubility, and are now widely employed in the magic angle spinning (MAS) DNP-NMR of proteins,1-6,33 including a recent example of in situ study of the bacterial type IV secretion system core complex.7 
While the magnitude of the DNP enhancement is one of the most important factors contributing to the absolute sensitivity of DNP-NMR measurements, many authors have pointed out to the significance of other parameters that may limit the total gain in the signal intensity.34,35 One factor is related to the necessity of achieving a homogeneous distribution of the polarizing agents that are exogenously added to a typically diamagnetic sample. Since all but a very few DNP experiments with protein samples are carried out in aqueous solutions and at below-freezing temperatures, a glass-forming solvent such as glycerol must be added to achieve a homogeneous distribution of the polarizing agents and prevent the formation of ice crystals, that is, to form a so-called glassy matrix.36 The necessity to use some large amounts of glycerol (10-20 mM concentration) effectively reduces the amount of protein in such a sample. For example, in the experiments with TOTAPOL described below, the incorporation of 60% glycerol leads to an approximately fourfold decrease in the maximally attainable protein concentration due to inefficient pelleting, thus, proportionally reducing the effective filling factor and the resultant NMR signal.
A number of alternative “matrix-free” sample preparation approaches have been described in the literature. In the first demonstration of DNP from covalently attached radicals, Miller, Griffin and co-workers have employed an endogenously present stable flavin mononucleotide radical, semiquinone, to enhance the NMR signal of flavodoxin.37 Bodenhausen et al. have covalently attached TOTAPOL to the C-terminal amino acid of a decapeptide through the ester bond.38 McDermott et al. reported on DNP enhancement from a “pseudo-biradical” formed upon the dimerization of gramicidin labeled by monoradicals at the dimer interface.39 De Paëpe et al. have taken advantage of the high partitioning of TOTAPOL within microcrystalline cellulose35 as well as its strong binding affinity to cell wall polymers (peptidoglycan)40 to obtain DNP-enhanced NMR spectra of the biopolymers constituting plant cell walls. DNP of phospholipid-embedded peptides from a mixture of two different monoradical-labeled lipids has been demonstrated by Long et al.,41 while, De Paëpe and collaborators have demonstrated the DNP of lipids in liposomes from a biradical functionalized with an acyl chain to provide for preferential partitioning into the lipid bilayer.42 
There remains a need for new and improved agents for dynamic nuclear polarization NMR studies of a range of analytes.